1. Field of the Invention
The invention relates to a species-specific DNA-DNA hybridization probe which is prepared using an isolated whole chromosome of a lower eukaryote as template. The probe is useful for the direction of conspecificity in lower eukaryotes.
2. Description of the Art
Identification of lower eukaryotic organisms is done by phenotypic characteristics. Using the genotype is a more stable identification method.
Yeast identification is a difficult yet needed scientific task. Accurate identification is required for epidemiology and identification of yeast-caused diseases and in the food industry where yeasts are a desired part of a given technology or cause spoilage.
Traditional technique for yeast identification is to perform morphological and physiological tests. These tests identify the cell shape and size as well as other characteristics. The physiological tests are based upon the ability of the organism to use certain chemicals and to grow in their presence or under anaerobic conditions to ferment them. In the two most universally used and thorough identification schemes, 50 to 100 tests are performed (J. A. Barnett et al., Yeasts: Characteristics and Identification, Cambridge University Press, 1983; N. J. W. Kreger van Rij (ed.), Yeasts--A Taxonomic Study, 3rd ed., Elsevier Science Publishers B.V., Amsterdam, in 1984). This testing can take up to six weeks. Computer programs have been developed to aid in the process of comparing test results with database entries for the almost 600 known species (J. A. Barnett et al., Yeast Identification Program, Cambridge University Press, Cambridge; American Society for Microbiology Computer User Group, 1989, Computer-Assisted Identification of Microorganisms, ASM, Washington, D.C.; T. Deak, 1990, Yeast-ID, University of Horticulture and Food Industry, Budapest). However, expertise on interpretation of the results is needed. Because of the amount of material required, the complexity of the tests and interpretation of the results, these tests are only done in specialized yeast laboratories or research laboratories.
Several commercial test kits for medically important yeasts have been developed and marketed. They are based upon the above mentioned reactions and are useful for disease identification. The number of medically important yeast species is limited; there are only about 50. Thus, it is relatively easy to create useful diagnostic kits for medical use. Candida albicans, the most important medical yeast is very often identified simply by germ tube formation. In the food industry almost 200 yeast species may occur, making identification more complex. No commercial test kits have been developed for this group of yeasts.
A newer development in the field of yeast identification was the reduction of the number of required tests (T. Deak, in A. D. King et al. (ed.), Methods for the Mycological Examination of Food, Plenum Publishing Co., New York, 1986; T. Deak et al., Journal of Food Protection 50: 243-264, 1987). The scheme was developed based upon a fairly even division of food-borne yeast species by the tests in question. In its final form, the simplified scheme uses about 25 tests to separate the 200 yeast species important in foods. A computer program has been developed to aid in the interpretation of the results.
In yeast taxonomy species differentiation is based upon interfertility. However, the establishment of conspecificity among homothallic and asporogenous yeasts and among industrially important, mostly aneuploids and/or polyploids, is rather hard on the basis of the phenotype. Comparisons between genotypic characteristics provide a more stabile classification. Thus, modern approaches analyze the G+C mol % (mol percent guanine and cytosine of DNA), the DNA homology by DNA-DNA re-association, and the DNA sequences coding for smaller rRNAs, etc.
DNA gel electrophoresis has been developed over the past several years. The development of pulsed field gel electrophoresis (PFGE) has permitted separation of relatively large pieces of DNA, even as large as chromosomes (Schwartz et al., Cell 37:67-75 (1984)). Contour clamped homogeneous electric field electrophoresis (CHEF) is one of the most recent developments of the basic PFGE (See Chu et al., Science 234: 1582-1585 (1986). CHEF allows for good resolution with preservation of sharp bands, straight lanes, and reproducible separation of DNA molecules up to 12-15 megabases. Some researchers have prepared probes using smaller than whole chromosome templates.